New geographical records for tick‐borne pathogens in ticks collected from cattle in Benin and Togo

Abstract Background Ticks are obligate hematophagous arthropods capable of transmitting a great variety of endemic and emerging pathogens causing diseases in animals and humans. Objectives The aim of this study was to investigate the presence of Bartonella spp., Rickettsia spp., Borrelia burgdorferi sensu lato (s.l.) and Anaplasma phagocytophilum in ticks collected from cattle in Benin and Togo. Methods Overall, 396 (148 males, 205 females and 43 nymphs) ticks were collected from cattle in 17 districts (Benin and Togo) between 2019 and 2020. Ticks were pooled into groups of 2–6 ticks per pool according to individual host, location, species and developmental stage. The DNA of each pool was extracted for molecular screening. Results PCR results revealed that 20 tick pools were positive for Bartonella spp. (Benin and Togo) and 23 tick pools positive for Rickettsia spp. (Benin), while all pools were negative for A. phagocytophilum and B. burgdorferi s.l. Sequence analysis of positive Rickettsia samples revealed the presence of Rickettsia aeschlimannii. Conclusions The present study highlights the presence of zoonotic agents in ticks collected from cattle in Benin and Togo. This information will raise awareness of tick‐borne diseases among physicians and veterinarians, stimulate further studies to monitor these pathogens, and advise on necessary measures to control the spread of these zoonoses.

monitor these pathogens, and advise on necessary measures to control the spread of these zoonoses.

K E Y W O R D S
bacteria, public health, Rickettsia aeschlimannii, tick, West Africa INTRODUCTION Ticks are known to be important disease vectors, capable of transmitting different pathogens such as protozoa, bacteria, and viruses to a wide range of hosts, including humans (Dantas-Torres et al., 2012). The distribution of tick-borne pathogens is influenced by a series of climatic and environmental factors, as well as density and diversity of the host species (Ogden, 2017;Wikel, 2018). Among the tick-borne pathogens, Rickettsia, Anaplasma or Bartonella species are of high importance in cattle (Kasaija et al., 2021). Rickettsia spp. are obligate intracellular alpha-proteobacteria, with a worldwide distribution and transmitted to humans and animals by various arthropod vectors, including ticks (Parola et al., 2013). They have a high host adaptability, and the infections are important causes of morbidity and mortality worldwide (Diop et al., 2019;Saravanan et al., 2020). Rickettsia spp. are considered emerging zoonotic pathogens that are responsible for conditions such as the spotted fevers and typhus (Bhengsri et al., 2016). The continuous identification of new species or genotypes of Rickettsia (Parola et al., 2013;Portillo et al., 2015) in ticks brings up new questions on their true diversity, ecology and biology and the role of climate change in their distribution (Dantas-Torres et al., 2012). This study provides information on these bacteria in West Africa, including Benin and Togo.
Bartonella spp. (Rhizobiales) are intracellular Gram-negative bacteria. About 30 Bartonella species were described in a great variety of domestic and wild mammals worldwide . Bartonella spp. cause endocarditis in humans and domestic animals, including cattle (Erol et al., 2013). Detection of Bartonella spp. in bats was reported from Ghana and Nigeria (Billeter et al., 2012;Kamani et al., 2014). It has also been detected in rodents and their ectoparasites in Nigeria (Kamani et al., 2013). Although several Bartonella spp. were identified in ticks, their vectorial competence is still under discussion (Telford & Wormser, 2010). Despite the potential public and veterinary health importance, no studies were conducted on these bacteria in Benin and Togo.
Borrelia burgdorferi sensu lato (s.l.) complex comprises more than 20 genospecies and was described worldwide in a great variety of animals and vectors including ticks (Margos et al., 2019). Borrelia burgdorferi s.l. is one of the most important tick-borne zoonotic agents in humans (Lefeuvre et al., 2020). They are maintained in nature by the interaction that exists with its vector ticks vertebrate reservoir hosts. Borrelia burgdorferi s.l. is transmitted by hard ticks and has been identified in Europe (Marchant et al., 2017), America (Scott et al., 2018) and Asia (Pukhovskaya et al., 2019), but few studies reported this zoonotic agent in Africa (Chitanga et al., 2014). While there are some reports of the presence of B. burgdorferi s.l. in Northern Africa (Elhelw et al., 2021), in Western Africa, mainly B. crocidurae and other species responsible for relapsing fever than Lyme borreliosis have been detected (Margos et al., 2019).
Anaplasma phagocytophilum was reported in several species of wild and domestic mammals worldwide, causing the human granulocytic anaplasmosis (HGA). In animals, it is responsible for reduction of milk production, abortion and also death (Stuen, 2007). Anaplasma phagocytophilum has been reported in various countries from Africa including Morocco, Zimbabwe, Tunisia, Algeria and South Africa (El Hamiani Khatat et al., 2021;Kelly et al., 2014;Nakayima et al., 2014). Currently, very few confirmed cases of A. phagocytophilum infection were reported in West Africa (Djiba et al., 2013).
In Africa, human tick-borne diseases are underestimated and tick bites in humans go unreported due to the lack of awareness, knowledge of the risk of TBDs and the failure of the epidemiological surveillance system (N'koué Sambiéni et al., 2015;Rapp, 2014;Tutin, 2000). However, accurate identification of pathogens circulating between wild and domestic animals, ticks, and humans in a region is essential to facilitate diagnosis and treatment regimens, which depend on the pathogen involved. The lack of epidemiological knowledge on tick-borne diseases induces confusion, with wrong diagnoses, and therefore, wrong treatment. The purpose of this study was to identify Bartonella spp., Rickettsia spp., B. burgdorferi s.l. and A. phagocytophilum in ticks collected from cattle in Benin and Togo (West Africa).

Molecular assessment
Ticks were pooled into groups of 2-6 ticks per pool according to individual host, location, species and developmental stage. The DNA of each pool was extracted using ISOLATE II Genomic DNA Kit The PCR amplifications were performed in 25 µl reaction volume, containing 12.5µ1 Green PCR Mastermix (Rovalab GmBH, Teltow, Germany), 6.5 µl of ultra-pure water, 1 µl (10 pmol/µl) of each primer (Table 1), and 4 µl of isolated DNA aliquot. One negative control (ultrapure water) as well as a positive control were included. For the nested PCR targeting the flaB gene of B. burgdorferi s.l. and 16S gene of A.

Sequencing
All sequences were analyzed and edited using Geneious® 4.85 software. Basic Local Alignments Tool (BLAST) analyses (https://blast.ncbi. nlm.nih.gov) were conducted to compare all the obtained sequences with the ones deposited in the GenBank™ database.  Barbour et al., 1996)

Sequencing
The gltA gene sequences carried out from Bartonella spp. obtained from two pools of R. microplus in Benin were closely related to a  (Tsai et al., 2010;Vayssier-Taussat et al., 2015). Our findings represent the first detection of Bartonella spp. in hard ticks in Benin and Togo. Bartonella spp. were previously also detected in rodents and ticks from Nigeria (Kamani et al., 2013).
This pathogen is implicated in several cases of fever in humans but also reported in several places in animals (Orkun et al., 2014b;Tosoni et al., 2016). The symptoms following an infection are similar to those of Mediterranean spotted fever caused by Rickettsia conorii (Rovery & Raoult, 2008). Raoult et al. (2002) reported cases of human infection caused by R. aeschlimannii in France.
New tick-borne pathogens infecting humans have increased in recent years, thus playing an important role in public health. In sub-Saharan Africa, R. aeschlimannii is known as a potentially important pathogen and was identified in humans, animals and ticks (Parola et al., 2013). In these regions, febrile illnesses in breeders, farmers and animal health professionals may be caused by rickettsiosis (Moumouni et al., 2016). Mediannikov et al. (2010) showed that tick-borne rickettsioses are among the causes of acute nonmalarial febrile diseases.
Traditionally, in West Africa, almost all febrile conditions are generally considered to be linked to malaria and are treated as such without further investigations, but other tick-borne pathogens might be involved.

CONCLUSION
This study reports the identification and distribution of pathogens in hard ticks collected from cattle in West Africa. These results suggest the need to include bartonellosis and rickettsiosis among the causes of febrile illnesses among breeders, para-veterinarians, veterinarians and travellers in West Africa. Doctors should also consider these tick-borne illnesses as a differential diagnostic with malaria. On the other hand, the role of domestic and wild animals in the epidemiology of diseases transmitted by ticks requires further investigation.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
No ethical approval was required, as this study does not involve clinical trials or experimental procedures. The cattle's are still alive and used for milk and meat production. This study did not involve endangered or protected species.